Our objective in this proposal is to apply immunologic techniques and principles to the study, detection, and therapy of breast cancer as part of an integrated laboratory and clinical effort combining immunologic, biochemical, molecular, and cell biologic approaches. In the past, the search for monoclonal antibody (mAb) targets in breast cancer focused on surface molecules of transformed mammary epithelium, but most of these epithelial markers show shortcomings related to variable expression in tumors (heterogeneity), presence in normal tissues or serum, or poor accessibility to circulating mAbs. Recently, new emphasis has been placed on alternative mAb targets in th abundant stromal compartment of breast carcinomas, including antigens of reactive stromal fibroblasts and of tumor capillary endothelial cells. Aim I focuses on the identification of new antigenic targets in breast cancer stoma, i.e. cell surface antigens of reactive stromal fibroblast and tumor capillaries. Prototypes for this category of tumor stromal antigens are the fibroblast activation protein, FAP, abundantly expressed in reactive fibroblasts of >90% of breast cancers; and endosialin, expressed in tumor capillaries of 60-70% of malignant solid tumors; additional stromal antigens are likely to be identified. The normal tissue distribution of tumor stromal antigens and their expression in benign, premalignant, and malignant breast lesions in patients in various stages of their disease will be documented by immunohistochemistry. Aim II will define the molecular structure of FAP, endosialin, and other antigens in breast cancer stroma, based on biochemical and gene cloning studies. For selected molecules, homologues in other species will be identified to design xenograft models of tumor stromal targeting. Aim III will explore stromal antigen function and mAb antigen interactions through cell biologic approaches. The newly derived serologic and DNA probes will be used to study the regulation of tumor stroma formation and activation, with emphasis on the role of soluble factors produced by malignant breast epithelial cells. The function of tumor stromal antigens may be discovered through sequence analysis or through blocking experiments with mAbs and antisense oligonucleotides in cultured cell models. The effects of first and second-generation mAbs and mAb-conjugates mediating immunologic and nonimmunologic effector functions, the fate of bound mAbs (internalization, processing), and imaging characteristics of radiolabeled mAbs in mouse xenograft models will also be examined. One of the main objectives is to develop a cocktail of humanized mAbs that can be used to target breast cancers, including existing mAbs against malignant epithelial cells and mAbs against reactive fibroblast or tumor capillary antigens. These mAbs may be used as adjuvant therapy in patients with minimal residual disease after surgery or chemotherapy. Pursuing these three aims should result in new concepts, new reagents, and new approaches to the detection and therapy of breast cancer.